Dna encoding modified heparin cofactor ii

ABSTRACT

A modified heparin cofactor II is disclosed, HCII (Leu 444  →Arg), which has substantially improved inhibitory activity against thrombin compared to native HCII or antithrombin.

BACKGROUND OF THE INVENTION

This invention relates to heparin cofactor II and, more particularly, toa modified heparin cofactor II that has substantially improvedinhibitory activity against thrombin compared to native heparin cofactorII or antithrombin.

The anticoagulant activities of glycosaminoglycans are mediated byantithrombin (AT) and heparin cofactor II (HCII), which are members ofthe `serpin` family of serine protease inhibitors [Tollefsen in Heparin:chemical and biological properties, clinical applications (eds. Lane andLindahl), Edward Arnold, London, 1989, pp. 257-273]. HCII inhibitsthrombin but has no activity against other proteases involved incoagulation or fibrinolysis [Parker and Tollefsen, J. Biol. Chem. 260,3501-3505 (1985)]. In contrast, AT inhibits all of the proteases of theintrinsic coagulation pathway as well as the fibrinolytic proteaseplasmin [Rosenberg in The molecular basis of blood diseases (eds.Stamatoyannopoulos et al.), W. B. Saunders Co., Philadelphia, 1987, pp.534-574]. Heparin and dermatan sulfate increase the rate of thrombininhibition by HCII˜1000-fold by providing a catalytic template to whichboth the inhibitor and the protease bind [Tollefsen et al., J. Biol.Chem. 258, 6713-6716 (1983); Griffith, Proc. Natl. Acad. Sci. USA 80,5460-5464 (1983)]. Although heparin catalyzes protease inhibition byboth HCII and AT, the anticoagulant effect of dermatan sulfate ismediated exclusively by HCII.

HCII is a 65,600 dalton plasma glycoprotein. Using conventionalrecombinant DNA gene splicing methods, the cDNA sequence andcorresponding amino acid sequence of HCII were determined and theprotein was expressed in E. coli by Blinder et al., Biochemistry 27,752-759 (1988). The cDNA consisted of 2215 base pairs (bp), including anopen-reading frame of 1525 bp, a stop codon, a 3'-noncoding region of654 bp, and a poly(A) tail. The deduced amino acid sequence contained asignal peptide of 19 amino acid residues and a native protein of 480amino acids. The published cDNA and protein sequences are also shownhereinbelow.

HCII forms a stable 1:1 complex with thrombin or chymotrypsin in whichthe protease is inactive. During complex formation, both thrombin andchymotrypsin attack the reactive site Leu₄₄₄ -Ser₄₄₅ peptide bond(designated P1-P1') near the C-terminal end of HCII [Church et al.,Proc. Natl. Acad. Sci. USA 82, 6431-6434 (1985); Griffith et al., J.Biol. Chem. 260, 2218-2225 (1985); Griffith et al., Biochemistry 24,6777-6782 (1985)]. The resulting complex does not dissociate when heatedat 100° C. with sodium dodecyl sulfate, suggesting that under theseconditions the two proteins become linked by a covalent bond. The bondis presumed to be an ester linkage between the serine hydroxyl group inthe active site of the protease and the carbonyl group of Leu₄₄₄ inHCII. Therefore, HCII can be thought of as a pseudosubstrate for theseproteases.

The P1 residue in the reactive site of a `serpin` appears to play amajor role in determining the relative rates of inhibition of variousproteases [Carrell and Travis, TIBS 10, 20-24 (1985)]. Evidence for thiswas derived from analysis of the variant α1-antitrypsin Pittsburgh,which was discovered in a child who had a fatal bleeding disorder. Thevariant contained an arginine residue in place of Met₃₅₈ at the P1position of the inhibitor [Owen et al., N. Engl. J. Med. 309, 694-698(1983)]. This substitution resulted in markedly increased rates ofinhibition of several coagulation proteases, including thrombin, factorXa, factor XIa, and kallikrein, which cleave Arg-X peptide bonds intheir natural substrates [Schapira et al., J. Clin. Invest. 77, 635-637(1986); Scott et al., Ibid. 77, 631-634 (1986); Travis et al., Biol.Chem. Hoppe-Seyler 367, 853-859 (1986)]. A reciprocal decrease in therate of inhibition of neutrophil elastase, the principal target proteaseof native α1-antitrypsin, was also noted.

Heparin is commonly employed in the prophylaxis and treatment of venousthrombosis and pulmonary embolism, but its use is sometimes complicatedby severe bleeding or thrombocytopenia. Therefore, alternatives toheparin for short-term anticoagulation would be desirable. In thisregard, a modified HCII which could inhibit thrombin rapidly in theabsence of heparin would have significant value and be useful for thetreatment of thrombotic disorders.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention a novel modified HCII has beendeveloped which has substantially improved inhibitory activity againstthrombin compared to native HCII or antithrombin. These improved resultsof about 100-fold increased inhibitory activity were unexpected in viewof the single site mutation of the native HCII which characterizes thenovel modified HCII. For convenience, the modified HCII of thisinvention can be represented as HCII(Leu₄₄₄ →Arg). In this mutation, theleucine residue at position 444 is replaced by a positively chargedarginine in the published 480 amino acid sequence of native HCII.

The modified HCII(Leu₄₄₄ →Arg) of this invention inhibited thrombin at a98-fold higher rate (k₂ =6.2×10⁶ M⁻¹ min⁻¹) than native HCII (k₂=6.3×10⁴ M⁻¹ min⁻¹). Dermatan sulfate accelerated thrombin inhibition byboth the native and modified forms of HCII, but the maximum rateconstant in the presence of dermatan sulfate was only 2-fold higher forHCII(Leu₄₄₄ →Arg)(k₂ =5.3×10⁸ M⁻¹ min⁻¹) than for native HCII (k₂=2.2×10⁸ M⁻¹ min⁻¹). Heparin was less effective than dermatan sulfate instimulating both forms of HCII. Factor Xa and kallikrein were inhibitedmore rapidly and chymotrypsin more slowly by HCII(Leu₄₄₄ →Arg) than bynative HCII. The rapid rate of inhibition of thrombin by HCII(Leu₄₄₄→Arg) in the absence of heparin or dermatan sulfate suggests that thisvariant may be useful as a therapeutic agent.

The single site mutation of HCII as defined herein is illustrated in arecombinant HCII (rHCII). However, the amino acid sequence of rHCII isidentical to that of HCII purified from human plasma. Thus, as definedherein the single site mutation HCII(Leu₄₄₄ →Arg) can be made on arecombinant HCII or a synthetically produced HCII.

Conventional recombinant DNA procedures can be used to prepare the novelHCII mutant of this invention. The starting material can be aconventional DNA construct or vector comprising a DNA sequence encodingfor the entire sequence of native HCII. The reactive site mutantrHCII(Leu₄₄₄ →Arg) is then prepared by replacing the codon for leucineat site 444 with the codon for arginine.

Thus, the DNA polymer can be prepared by the enzymatic ligation ofchemically synthesized fragments. The DNA polymer which encodes themodified HCII may be prepared by site-directed mutagenesis of the cDNAwhich codes for HCII by conventional methods such as those described byWinter et al., Nature 299, 756-758 (1982) or Zoller and Smith, Nucl.Acids Res. 10, 6487-6500 (1982). Expression of the modified HCII can becarried out by conventional methods.

A suitable starting material for preparing the HCII(Leu₄₄₄ →Arg) of thisinvention is the known plasmid expression vector pMON-5840 as describedby Blinder et al., J. Biol. Chem. 264, 5128-5133 (1989). This plasmid isa variant of pMON5527 which, in addition to an irrelevant sequence,contains the recA promoter (P_(rec) A) and the ribosome-binding site, T7phage gene 10 leader RNA (G10-L RBS), and is suitable for enhancedexpression of foreign genes in E. coli, as further described by Olins etal., Gene 73, 227-235 (1988); Olins and Rangwala, J. Biol. Chem. 264,16973-16976 (1989). The recA promoter in pMON-5840 is derived as theHpaII fragment and corresponds to nucleotides 63-210 of the sequencepublished by Horii et al., Proc. Natl. Acad. Sci. USA 77, 313-317(1980). The cDNA for HCII(Leu₄₄₄ →Arg) can be ligated into the parentvector which can then be used for expression of the rHCII(Le₄₄₄ →Arg) byconventional procedures, e.g. in E. coli JM101 cells.

DETAILED DESCRIPTION OF THE INVENTION

While the specification concludes with claims particularly pointing outand distinctly claiming the subject matter regarded as forming thepresent invention, it is believed that the invention will be betterunderstood from the following detailed description of preferredembodiments of the invention taken in conjuction with the appendeddrawings in which:

FIG. 1 is a schematic diagram of the expression vector pMON-HCII. HCIIcDNA (shaded rectangle) was altered in M13 to remove a SalI site andcreate a BstEII site. Cassette mutagenesis between BstEII and SalI wasperformed to create the reactive site mutant pMON-HCII(Leu₄₄₄ →Arg). Theopen rectangle denotes the 3'-untranslated sequence from the HCII cDNA.The RecA promoter (P recA) and G10L translational control element,derived from the T7 gene, are indicated, along with the deletion of aSalI site in the parent vector pMON-5840. The codons and amino acids atthe translation initiation site and in the reactive site cassette areshown.

FIG. 2 shows the immunoblot of pMON-HCII, pMON-HCII(Leu₄₄₄ →Arg) andpMON-5840 lysates with anti-HCII antibody. pMON-HCII, pMON-HCII(Leu₄₄₄→Arg) and pMON-5840 were expressed in E. coli and subjected toheparin-Sepharose® chromatography, as described below under"Procedures." The partially purified rHCII, rHCII(Leu₄₄₄ →Arg) andcontrol lysate were immunoblotted and probed with ¹²⁵ I-labelled rabbitanti-HCII antibodies. An autoradiograph of the immunoblot is shown. Theamount (μl) of partially purified lysate loaded in each lane isindicated. Molecular weight markers are shown on the right hand side.

FIG. 3 (parts A-D) is a graphical representation which shows theinhibition of thrombin, chymotrypsin, factor Xa and kallikrein by rHCIIand rHCII(Leu₄₄₄ →Arg). Heparin-Sepharose column eluates (90 μl) ofrHCII (142 nM), rHCII(Leu₄₄₄ →Arg) (76 nM) or pMON-5840 control wereincubated with thrombin (15 nM), chymotrypsin (21 nM), factor Xa (16nM), or kallikrein (10 nM) for the indicated amount of time at thestated final concentrations. The % protease activity was calculated fromthe ratio of final to initial protease activity, determined by addingthe appropriate chromogenic substrate and measuring Δ405/min asdescribed below under "Procedures." , Control; ◯, Native rHCII; ,rHCII(Leu₄₄₄ →Arg).

FIG. 4 is a graphical representation which shows the effect ofglycosaminoglycan concentration on the second-order rate constant ofthrombin inhibition by rHCII and rHCII(Leu₄₄₄ →Arg). Second-order rateconstants were determined by incubating heparin-Sepharose column eluates(90 μl) of rHCII (142 nM) or rHCII(Leu₄₄₄ →Arg) (76 nM) with thrombin(15 nM) and either heparin or dermatan sulfate at the indicated finalconcentrations. The rate constants were calculated as described belowunder "Procedures." After times ranging from 5 seconds to 5 minutesremaining thrombin activity was determined by adding Chromozym TH andmeasuring Δ405/min. The rate constants with dermatan sulfate are theaverage of three similar tests. The heparin-stimulated rate constantsare from a single determination. ◯, rHCII plus heparin; , rHCII(Leu₄₄₄→Arg) plus heparin; □,rHCII plus dermatan sulfate; , rHCII(Leu₄₄₄ →Arg)plus dermatan sulfate.

In order to illustrate the invention in greater detail, the followingexemplary laboratory preparative work was carried out and the resultsobtained as described and shown in Table 1 and the accompanyingdrawings.

EXAMPLES Procedures

Materials--Chromogenic substrates were purchased from the followingsources: tosyl-Gly-Pro-Arg-p-nitroanilide (Chromozym TH), BoehringerMannheim; N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilde, Sigma;methoxycarbonyl cyclohexylglycyl-Gly-Arg-p-nitroanilde (SpectrozymeFXa), American Diagnostica; and Pro-Phe-Arg-p-nitroanilide (S-2302),KabiVitrum. Thrombin and HCII derived from human plasma were preparedand assayed by conventional procedures as previously described byBlinder et al., J. Biol. Chem. 264, 5128-5133 (1989). Human coagulationfactor X_(a) was provided by Dr. George Broze, Jr., WashingtonUniversity, St. Louis, Mo. Human chymotrypsin and plasma kallikrein werepurchased from Sigma. Affinity purified rabbit anti-HCII antibodies wereprepared and labeled with Na[¹²⁵ I] by conventional procedure asdescribed previously by Blinder et al., Biochemistry 27, 752-759 (1988).Bovine lung heparin and porcine skin dermatan sulfate were purchasedfrom Sigma; the dermatan sulfate was treated with nitrous acid to removecontaminating heparin prior to use [Teien et al., Thromb. Res. 8,859-867 (1976)]. Heparin-Sepharose was prepared by commonly knownprocedure as described previously by Tollefsen et al., J. Biol. Chem.257, 2162-2169 (1982).

The reagents used for oligonucleotide directed mutagenesis werepurchased from Amersham. Restriction enzymes were obtained from Promega,Amersham, New England Biolabs, and Bethesda Research Laboratories.Reagents for the Tag polymerase chain reaction were purchased fromPerkin Elmer Cetus. DNA sequencing was performed using T7 DNA polymerasefrom U.S. Biochemical Corporation. Deoxyadenosine [α³⁵S]thiotriphosphate([α³⁵ S]dATP) was obtained from Dupont-New EnglandNuclear. The Protein Chemistry Facility of Washington University, St.Louis, Mo., provided the synthetic oligonucleotides.

Construction of the pMON-HCII expression vector--Previously, thePstI-HindIII fragment of the HCII cDNA was inserted into pMON-5840 toyield a construct in which the N-terminal 18 amino acid residues ofmature plasma HCII were replaced by the sequence Met-Ala- as describedby Blinder et al., J. Biol. Chem. 264, 5128-5133 (1989). Although theresulting inhibitor was active by several criteria, it was desired toobtain a full-length rHCII for the purposes of the present invention. Inaddition, it was decided to construct unique SalI and BstEII restrictionsites for cassette mutagenesis of the reactive site. The Sall site inthe parent vector pMON-5840 was deleted to form pMON-5840(ΔSalI) bydigestion with SalI, treatment with mung bean exonuclease, andreligation. A full-length cDNA for HCII was isolated previously from ahuman fetal liver library in λgt11 as described by Blinder et al.,Biochemistry 27, 752-759 (1988). Oligonucleotide-directed mutagenesis ofthe cDNA was performed in M13mp18 phage vector containing the non-codingstrand by the published method of Nakamaye and Eckstein, Nucl. AcidsRev. 14, 9679-9698 (1986). The codon for Val₇₇ was altered (GTC→GTT) toremove a SalI site at that position, and the codon for Thr₄₃₄ wasaltered (ACT→ACG) to construct a BstEII site. Using the polymerase chainreaction with appropriate primers [Saiki et al., Science 239, 487-491(1988)], an NcoI site (CCATGG) was incorporated into the cDNA so thatthe ATG of the NcoI site immediately preceded the codon for Gly₁ ofmature plasma HCII. The amplified cDNA was then ligated intopMON-5840(ΔSalI). The final construct, designated pMON-HCII, isdiagrammed in FIG. 1.

Cassette mutagenesis of pMON-HCII--pMON-HCII was digested with SalI andBstEII to remove a fragment of DNA containing the codons for Val₄₃₆-Val₄₅₂, which span the reactive site. The vector was then treated withalkaline phosphatase. Complementary oligonucleotides (48- and 49-mers)containing the codon for arginine at position 444 were phosphorylatedwith T4 kinase, preannealed, and then ligated to the vector. E. colistrain JM101 cells were transformed with the vector, and colonies wereselected for ampicillin resistance. The sequence of the final plasmidwas verified using the dideoxy chain termination method of Sanger etal., Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977).

Expression and quantification of native rHCII and rHCII(Leu₄₄₄→Arg)--For each rHCII preparation, transformed E. coli were grown to anoptical density (550 nm) of 1.0-1.2 in 500 ml of medium and expresiionwas induced with nalidixic acid according to Li et al., J. Biol. Chem.262, 13773-13779 (1987). The cells were then sedimented, rinsed once in0.05M NaCl, 0.05M Tris-HCl, pH 7.5, resuspended in 25 ml of the samebuffer, and lysed by sonication [Blinder et al., Biochemistry 27,752-759 (1988)]. To partially purify the rHCII, the cell lysate wasapplied to a 30 ml column of heparin-Sepharose equilibrated with thesame buffer. After the column was washed, the rHCII was eluted with 1.0MNaCl, 0.05M Tris-HCl, pH 7.5. The eluate was dialyzed against 0.05MNaCl, 0.05M Tris-HCl, pH 7.5, and stored frozen.

The concentration of rHCII was determined by a slot blot immunoassay.Various amounts of the partially purified rHCII were blotted ontonitrocellulose, and rHCII was detected with ¹²⁵ I-labeled anti-HCIIantibodies as described previously by Blinder et al., Ibid. Afterautoradiography, the bands were scanned with a densitometer and theareas under each peak were determined (LKB Ultroscan). A standard curveconstructed with known amounts of plasma HCII was linear from 2.5 to 30ng. The standard deviation of multiple determinations of plasma HCIIusing this method was 20%.

Inhibition of proteases by rHCII--Protease inhibition by rHCII wasdetermined by incubating 90 μl of the partially purified rHCII (or thecorresponding heparin-Sepharose fractions of a control lysate from cellstransformed with pMON-5840) with 5 μl of the protease and 5 μl of eitherglycosaminoglycan or water in a disposable polystyrene cuvette at roomtemperature. Final concentrations of the proteases were as follows:thrombin, 15 nM; chymotrypsin, 21 nM; factor Xa, 16 nM; and kallikrein,10 nM. After a specified period of time from 5 seconds (s) to 150 min,300-500 μl of the appropriate chromogenic substrate was added, and theabsorbance at 405 nm was recorded continuously for 100 s. The rate ofchange of absorbance was proportional to the concentration of activeprotease remaining in the incubation. The amounts added andconcentrations of each substrate were as follows: Chromozym TH(thrombin), 500 μl, 0.1 mM; N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilde(chymovtrypsin), 300 μl, 0.5 mM; Spectrozyme FX_(a) (factor Xa), 400 μl,0.125 mM; and S-2302 (kallikrein), 300 μl, 0.4 mM.

Results

Expression vectors for rHCII--A diagram of the HCII expression vector(pMON-HCII) is shown in FIG. 1. The mRNA produced by this vectorcontains an initiator Met codon followed by the nucleotide sequenceencoding the mature polypeptide (i.e., minus signal peptide) of nativeHCII. Edman degradation of rHCII purified from a cell lysate revealedthat the sequence begins with Gly:, indicating that the initiating Metresidue is removed during biosynthesis in E. coli. Thus, the amino acidsequence of native rHCII is identical to that of HCII purified fromhuman plasma. The reactive site mutant rHCII(Leu₄₄₄ →Arg) was preparedby insertion of the appropriate synthetic oligonucleotide cassette intopMON-HCII digested with BstEII and SalI as indicated in FIG. 1.

Immunoblot and quantification of rHCII--FIG. 2 shows the autoradiographof an immunoblot of lysates of E. coli transformed with pMON-5840(parent vector lacking the HCII cDNA), pMON-HCII, or pMON-HCII(Leu₄₄₄→Arg). Each lysate was subjected to heparin-Sepharose chromatography asdescribed above under "Procedures" prior to immunoblotting. Theimmunoblot was probed with ¹²⁵ I-labeled rabbit anti-HCII antibodies.The figure indicates that the rHCII expressed by pMON-HCII(Leu₄₄₄ →Arg)is identical in size to that expressed by pMON-HCII and has an apparentmolecular weight of˜60,000. A minor band (MW=43,000) containing 5-10% ofthe HCII antigen is also present in both lysates and may represent adegraded form of the protein. No HCII antigen is present in a lysate ofE. coli transformed with pMON-5840. The level of expression of rHCII wasdetermined by densitometry of immunoblots standardized with knownamounts of purified plasma HCII. The rHCII concentration in thepMON-HCII and pMON-HCII(Leu₄₄₄ →Arg) lysates (after heparin-Sepharosechromatography) were 158 nM and 84 nM, respectively.

Protease inhibition by rHCII in the absence of a glycosaminoglycan--Thetime courses of inhibition of thrombin, factor Xa, kallikrein, andchymotrypsin by native rHCII(Leu₄₄₄ →Arg) in the absence of aglycosaminoglycan are indicated in FIG. 3. The incubations wereperformed under pseudo-first-order conditions, such that the initialconcentration of rHCII was greater than the concentration of protease.The pseudo-first-order rate constants (k₁) for protease inhibition weredetermined by fitting the data to the following equation: k₁ t=ln([P]₀/[P]_(t)), in which [P]₀ =initial protease activity and [P]_(t)=protease activity at time=t. The second-order rate constants (k₂) werethen calculated by dividing k₁ by the initial rHCII concentration. Therate constants are summarized in Table 1. It was observed thatrHCII(Leu₄₄₄ →Arg) inhibits thrombin almost 100 times more rapidly thannative rHCII in the absence of a glycosaminoglycan. Furthermore,rHCII(Leu₄₄₄ →Arg) inhibits factor Xa and kallikrein, while inhibitionof these proteases by native rHCII is undetectable. In contrast tonative rHCII, rHCII(Leu₄₄₄ →Arg) does not inhibit chymotrypsin at adetectable rate.

Inhibition of thrombin by rHCII in the presence of dermatan sulfate orheparin--The time course of inhibition of thrombin was determined in thepresence of various concentrations of dermatan sulfate or heparin. Thesecond-order rate constants derived from these tests are shown in FIG.4. Maximum rate constants were observed at 25-100 μg/ml of eitherglycosaminoglycan (Table 1). Dermatan sulfate produces approximately a3500-fold increase in the rate constant for thrombin inhibition bynative rHCII but has a much smaller effect on the rate constant obtainedwith rHCII(Leu₄₄₄ →Arg). Thus, rHCII(Leu₄₄₄ →Arg) inhibits thrombin onlyabout twice as fast as native rHCII in the presence of dermatan sulfate.Heparin increases the rate constant for thrombin inhibition by nativerHCII about 40-fold but has a minimal effect on the rate constantobtained with rHCII(Leu₄₄₄ →Arg).

Inhibition of chymotrypsin, factor Xa and kallikrein by rHCII in thepresence of dermatan sulfate or heparin--Dermatan sulfate does notsignificantly increase the rate of inhibition of chymotrypsin by nativerHCII, nor does it increase the rate of inhibition of factor Xa orkallikrein by rHCII(Leu₄₄₄ →Arg) (Table 1). Heparin (100 μg/ml) appearsto protect factor Xa and kallikrein from inhibition by rHCII(Leu₄₄₄→Arg).

                                      TABLE 1                                     __________________________________________________________________________    Second-order rate constants (k.sub.2) for inhibition of proteases by          rHCII                                                                                native rHCII        rHCII(Leu.sub.444 →Arg)                                   + Dermatan          + Dermatan                                  Protease                                                                             No GAG Sulfate                                                                              + Heparin                                                                           No GAG Sulfate                                                                              + Heparin                            __________________________________________________________________________           k.sub.2 (M.sup.-1 min.sup.-1)                                          Thrombin                                                                             6.3 × 10.sup.4 a                                                               2.2 × 10.sup.8 b                                                               2.4 × 10.sup.6 c                                                              6.2 × 10.sup.6 a                                                               5.3 × 10.sup.8 b                                                               1.6 × 10.sup.7 c               Chymotrypsin                                                                         2.2 × 10.sup.5 a                                                               3.7 × 10.sup.5 c                                                               2.4 × 10.sup.5 c                                                              <1 × 10.sup.4 b                                                                <1 × 10.sup.4 c                                                                N.D.                                 Factor Xa                                                                            <1 × 10.sup.3 b                                                                <1 × 10.sup.3 b                                                                N.D.  4.4 × 10.sup.4 c                                                               6.2 × 10.sup.4 b                                                               2.1 × 10.sup.4 b               Kallikrein                                                                           <3 × 10.sup.3 c                                                                N.D.   N.D.  1.3 × 10.sup.5 c                                                               1.4 × 10.sup.5 c                                                               1.0 × 10.sup.4                 __________________________________________________________________________                                             c                                     The rate constants were determined as described above under "Procedures"      and are derived from the data in FIGS. 2 and 3. The maximum rate constant     obtained in the presence of 25-100 μg/ml of dermatan sulfate or hepari     are given.                                                                    GAG, glycosaminoglycan.                                                       N.D., not determined.                                                         .sup.a Average of three determinations.                                       .sup.b Average of two determinations.                                         .sup.c Single determination.                                                  ##STR1##

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. It is intended that all such other examplesbe included within the scope of the appended claims.

What is claimed is:
 1. An isolated DNA sequence encoding for humanheparin cofactor II with an amino acid substitution of arginine atposition 444 for a leucine.